Apparatus and Method for Separating and Isolating Components of a Biological Fluid

ABSTRACT

A device for separating and isolating components of a biological fluid comprising a container for containing the fluid to be processed, a tube cap assembly for dosing the container while providing filling and extraction communication therewith, a float assembly disposed within the container for funneling and controlling biological fluid flow into an inverted domed shaped isolation chamber within the float and controlling the biological fluid flow out of the isolation chamber for effecting an encapsulation or a sealed isolation of at least one component or fraction of the biological fluid flow within the isolation chamber during a centrifugation process. The device further comprising a flexible tube for connecting an extraction passageway disposed within the float assembly and an extraction valve of the tube cap assembly for allowing extraction of at least the one component or fraction encapsulated or isolated within the chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 USC §120, this application is a continuation patentapplication of U.S. patent application Ser. No. 13/506,145, filed Mar.30, 2012, currently pending, and which is incorporated herein byreference in its entirety and which is a continuation patent applicationof U.S. application Ser. No. 12/315,722, filed Dec. 4, 2008, and issuedMay 15, 2012 as U.S. Pat. No. 8,177,072, and which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to devices and methods for separatingbiological fluids into components having different densities and, inparticular, to an apparatus and method for receiving a biological fluidsample, separating components of the biological fluid sample whileisolating at least one target component from non-target components ofthe biological fluid sample based upon fluid component densitydifferences, and extracting at least one isolated target component inpreparation for at least one diagnostic or therapeutic application. Thisinvention is particularly useful in the centrifugal separation of bloodand bone marrow into components.

BACKGROUND OF THE INVENTION

It is known to separate biological fluids, such as aspirated bone marrowor peripheral blood, into their component parts, fractions, phases, orconstituent layers by centrifugation. It is also known to providemechanical devices comprised of a tube which houses a solid separatorwhich, when actuated by centrifugal force, allows biological fluid toflow through or around the piston based on differing relative densitiesthereby separating the biological fluid into a one or more componentparts above and one or more component parts below the solid separator.For example, when the biological fluid within the tube is blood, thecentrifugation process results in a high density layer of red bloodcells below the solid separator, a low density layer of plasma above thesolid separator, and a huffy coat layer which defines an intermediatedensity layer or third fraction above the solid separator and below thelow density layer of plasma.

One of the earliest solid separators was disclosed in U.S. Pat. No.3,508,653, issued Apr. 28, 1970 to Coleman. That device was a rubber orother elastomeric cylinder. A major problem with that device was theinability to maintain a seal because it is costly to maintain theprecise inner diameter of the test tube when mass produced. A subsequentsolid separator development is disclosed in U.S. Pat. No. 3,814,248,issued Jun. 4, 1974 to Lawhead. Next, U.S. Pat. No. 3,779.383, issuedDec. 18, 1973 to Ayres disclosed a device in which the bloodintroduction end of the tube is opposite to the movable separator end ofthe tube, and abutting an impenetrable rubber closure. Following Ayres,U.S. Pat. No. 3,931,018, issued Jan. 6, 1976 to North, Jr. disclosed asolid separator for use in separation of blood serum and blood plasmausing centrifugal force that must be inserted into the blood collectiontube after blood collection.

In a patent to Levine, et al. (U.S. Pat. No. 4,159,896, issued Jul. 3,1979) centrifugally motivated solid separator device is disclosed inwhich a cylindrical float is disposed inside of a tube, which float hasan accurately controlled outside diameter so as to fit snugly in thetube bore under static conditions. When used in harvesting blood cellsthe float is formed with an axial through bore which receives andexpands the white cell and platelet layers in the blood sample aftercentrifugation thereof. The disclosed float was made from a plasticmaterial having a specific gravity that causes it to float in the packedred cells after centrifugation of the blood sample in the tube.

In another patent to Levine, et al, (U.S. Pat. No. 5,393,674, issuedFeb. 28, 1995) a clear plastic tube large enough to process 1 ml ofblood and equipped with a cylindrical float and filled with an inert gasat low pressure is disclosed. The float contains a through bore, andprior to centrifugation, is held fixably at an initial location by tightcontact between the exterior of the float and the interior wall of thetube. Unlike the inventions of Coleman, which contain pistons (or buoys)with no through bore, the Levine float relocates, under centrifugation,to a new position determined by its density relative to the density ofthe blood fractions as a result of the shrinkage of its diameter due tothe longitudinal elongation (and subsequent lateral narrowing) of thefloat body that results from the substantial gravity gradient thatoccurs from the top to the bottom of the float. This substantial G forcegradient (several thousand Gs) causes the float to elongate and narrowjust as a rubber tube elongates and narrows when pulled from both ends.This space between the exterior of the float and the interior of thetube that develops during centrifugation provides the freedom ofmovement of the float consequent with the motion of the blood componentsto their new location determined by their density relative to the float.Levine does not posit, but it is assumed that some of the redistributingblood components also travel through the bore during centrifugation butsince the top and bottom of the through bore are not closed, any cellsand platelets that wind up there following centrifugation are easilyinfiltrated by the red cells and plasma during normal postcentrifugation handling. Designed predominately as a diagnostic toolthat proceeds through the visual examination of the cells that at leasttemporarily occupy the through bore right after centrifugation, Levinealso discloses the possibility of extracting these cells with a syringeneedle for additional diagnostic examination. This method of extractionnecessarily is inefficient as a means of cell recovery as the intrudingneedle necessarily relocates the target cells above and below thethrough bore as it is inserted.

Hence, these known mechanical devices are generally capable ofseparating biological fluids into component parts or fractions; however,these devices are not very precise thereby resulting in inefficientseparation of the biological fluid into component parts or fractionsbecause of the substantial comingling of the separated fractions.Additionally, these known mechanical devices fail to provide a simple orefficient method to extract a fraction other than the top fraction ofthe sample leading to low recoveries, especially of the clinicallyimportant buffy coat fraction.

It is also known to provide more complicated mechanical devices in anattempt to alleviate the above known problems. For example, the patentto Leach, et al. (U.S. Pat. No. 7,374,678, issued May 20, 2008) in afirst embodiment, discloses a device for separating a sample, such asblood, into a plurality of fractions. The device is comprised of aplunger (or second piston) which, prior to centrifugation, is retainedproximate a top end of a closed ended distortable tube duringcentrifugation and a first piston (or buoy) which is tightly fitted nearthe bottom of the closed ended distortable tube such that undercentrifugation with a sample of blood, the tube wall longitudinallycompresses and bows outward thereby allowing the buoy to move in adirection of the top of the tube lifted by a layer of red blood cells ofhigher density than the piston that has flowed downward between the buoyand the interior of the tube wall. After centrifugation, the tube wallreturns to its original dimension and traps this first piston at a newlocation coinciding with the interface position of a top plasma fractionand a bottom red blood fraction of the separated sample. On or near acollection face of this first piston (or buoy) is a third fraction whichincludes “a small, yet concentrated, amount of red blood cells, whiteblood cells, platelets, and a substantial portion of a buffy coat of theblood sample.” The device then employs a plunger (or second piston whichis manually pushed down into the tube from a location proximate the topend of the tube. The plunger for second piston) includes a valve whichallows the plasma to pass through the plunger to while the plunger islowered to a predetermined depth above the first piston set by a depthgauge which locates the plunger a distance away from the collection faceof the piston thereby defining a third fraction between a bottom face ofthe plunger (or second piston) and the collection face of the firstpiston. The extraction of the third fraction is accomplished via avacuum created on a tube extending between a collection valve disposedin the top of the tube and a bore extending from the top of the plungerand the bottom of the plunger.

Accordingly, this device relies on the imprecise longitudinalcompression and decompression of the tube wall in order to control theflow path between fractions and fails to contain the separated fractionsuntil after centrifugation stops and the tube wall returns to itsoriginal dimensions. Furthermore, the extraction of the third fractionrequires infiltration of the top plasma fraction. Hence, this recentlypatented device still fails to alleviate the problem of inefficientseparation of the biological fluid into component parts or fractions andthe comingling of the separated fractions.

In another embodiment, Leach, et al. discloses that the plunger (orsecond piston) is rigidly or slideably fitted with the first piston orbuoy such that the pair is tightly fitted within the closed endeddistortable tube wherein under centrifugation with a sample such ofblood, the tube wall bows outward thereby allowing the pair to move in adirection of the top of the tube while lifted by a high density layer ofred blood cells flowing downward between the pair and the interior ofthe tube wall. After centrifugation, the tube wall returns to itsoriginal dimension which grips the periphery of the first piston at aninterface position of a plasma fraction and a red blood fraction of theseparated sample. On or near a collection face of this first piston is“a small, yet concentrated, amount of red blood cells, white bloodcells, platelets, and a substantial portion of a huffy coat of the bloodsample.” The extraction of the intermediate (buffy coat) or thirdfraction is accomplished “by interconnecting a cannula or bored tubewith the connection portion of the buoy cylinder” and connecting anextraction syringe to the cannula for creating a vacuum to draw theintermediate or third fraction from the space between the first andsecond pistons. This embodiment describes only one centrifugation spin,and fails to alleviate the problem of inefficient separation of thebiological fluid into component parts or fractions and the comingling ofthe separated fractions. Furthermore, the extraction of a fraction otherthan the top fraction still requires the infiltration of at least oneother fraction than the desired fraction to he extracted. Moreover, thedevice relies on the imprecise longitudinal compression anddecompression of the tube wall in order to control the flow path betweenfractions and fails to contain the separated fractions untilcentrifugation stops and decompression of the tube wall is concluded.

Another problem associated with both embodiments of Leach, et al. isthat the collection face, trough, or sump of the buoy must be shallow tobe at a desired density level of the target buffy coat fraction and topreclude even further accumulation of reds cells with the target whitecells and platelets to be extracted. Thus, this shallow trough resultsin having the target white blood cells and platelets, come to rest onthe entire large surface area of the first piston on which the whiteblood cells and platelets tend to stick, which reduces the efficiency ofthe final collection step. A further problem associated with bothembodiments of Leach, et al. is the time consuming and laborious processof fitting and interconnecting multiple parts to the device in order toperform the extraction process.

In general, current processes for separating and extracting fractionsout of biological fluids require multiple steps that are both laboriousand time consuming and that result in poor recoveries of the targetwhite cells and platelets. Hence, it would be desirable to provide asimplified and more effective process so less time, labor, and trainingis required to do the procedure and fewer white cells and platelets arelost thereby providing a positive economic impact. A simplified processwould also allow it to be performed in an intra operative setting by anoperating room nurse, rather than a remote laboratory setting by atechnician so that a patient can be more rapidly treated and thepossibility of mixing up samples can be essentially eliminated. Processsimplification also has a direct correlation to process reproducibilitythat is also a problem with the known prior art.

Hence, the known prior art is problematic in a number of areas whichinclude a deficiency in the recovery efficiency of cells of interest(target cells), in the selectivity of separation for reducingcontamination or non-target cells from the target cell population, andin the multiple step, laborious, and time consuming extraction process.

Accordingly, there is a need to overcome the significant shortcomings ofthe known prior-art as delineated hereinabove.

BRIEF SUMMARY OF THE INVENTION

Accordingly, and in one aspect, an embodiment of the inventionameliorates or overcomes one or more of the significant shortcomings ofthe know prior art by providing a separation and isolation devicecomprising a float assembly slideably enveloped in an enclosurecontaining biological fluid for promoting fluid flow into and automaticself sealing of a chamber of the funnel float for isolating at least onecomponent or fraction of a biological fluid being processed.

In another aspect, an embodiment of the invention provides a steepsloped entry way to a self sealing isolation chamber so that componentssuch as cells that are flowing into the isolation chamber are precludedfrom sticking on the surface area of the sloped entry way.

In another aspect, an embodiment of the invention provides a floatcomprised of an upstream valve and a downstream valve located in acirculation path of fluid traversing through an isolation chamber of thefloat sealed by closure of the valves as a function of a firstdifferential pressure on the upstream valve and a second differentialpressure on the downstream valve for isolating a target component of abiological fluid within the float.

In another aspect, an embodiment of the invention provides a device forseparating and isolating components of a biological fluid withcentrifugation, the device comprising: an enclosure for containing abiological fluid having multiple components; a float slideably disposedwithin the enclosure and having an interior isolation chamber; a firstvalve means for allowing a flow of biological fluid into the interiorisolation chamber of the float as a function of a first pressuredifferential of biological fluid on the first valve means; and a secondvalve means for providing a flow of biological fluid out of the interiorisolation chamber of the float as a function of a second pressuredifferential of biological fluid on the second valve means wherein underdevice centrifugation the first valve means and the second valve meansinitially allow components of the biological fluid to flow through theinterior isolation chamber and subsequently close to seal the interiorisolation chamber as the function of the first pressure differential ofbiological fluid on the first valve means and as the function of thesecond pressure differential of biological fluid on the second valvemeans for isolating at least one target component of the biologicalfluid within the interior isolation chamber of the float.

In another aspect, an embodiment of the invention provides a device forseparating and isolating components of a biological fluid withcentrifugation, the device comprising: an enclosure comprising aninterior circumferential surface defining a chamber for containing abiological fluid having multiple components; a float slideably disposedwithin the enclosure and partitioning the enclosure into a lower volumezone and an upper volume zone; the float comprising an interiorisolation chamber defining an intermediate volume zone and an exteriorcircumferential surface circumferentially spaced from the interiorcircumferential surface of the enclosure for defining a circumferentialgap therebetween; a first valve means for opening and closingcommunication of biological fluid from the upper volume zone to theinterior isolation chamber within the float; and a second valve meansfor opening and closing communication of biological fluid from theinterior isolation chamber within the float to the lower volume zone ofthe container wherein under centrifugation the first valve means and thesecond valve means control at least one closed loop circulation ofbiological fluid into the interior isolation chamber from the uppervolume zone, out of the interior isolation chamber into the lower volumezone, out of the lower volume zone and through the circumferential gapto the upper volume zone, and back into the interior isolation chamberas a function of a first pressure differential of biological fluid onthe first valve means and as a function of a second pressuredifferential of biological fluid on the second valve means for isolatingat least one target component of the multiple component biological fluidwithin the interior isolation chamber of the float.

In another aspect, an embodiment of the invention provides a method forseparating a biological fluid having multiple comingled fractions usinga conventional centrifuge device, the steps comprising; centrifuging adevice containing a biological fluid having multiple comingled fractionsfor forming a first fraction, a second fraction and a third fraction ofthe biological fluid; and utilizing a valve means for controlling entryand exit of the biological fluid through an interior isolation chamberof a float of the device for isolating the third fraction within theinterior isolation chamber of the float during the centrifuging step.Additionally, and in one embodiment, the above method further comprisesa step of activating a magnetic stirring bar disposed within theinterior isolation chamber of the float for stirring the third fractionafter the centrifuging step. Furthermore, and in one embodiment, theabove method further comprises a step of harvesting the stirred thirdfraction of the biological fluid from the device by using an aperture inopen communication with the interior isolation chamber of the float.Moreover, and in one embodiment, the above method further comprises astep of coupling at least one weight to the float for selectively tuningthe density of the float to about 1.02 grams/cubic centimeter to about1.08 grams/cubic centimeter prior to the centrifuging step.

In a particular aspect, an embodiment of the invention provides a devicefor separating and isolating components of a biological fluid withcentrifugation, the device comprising: a container comprising a closedbottom end, an open top end, and a container sidewall extending betweenthe closed bottom end and the open top end, the sidewall having an innercircumferential surface defining a containing chamber extending along acentral longitudinal axis of the container; a cap for selectivelyclosing the open top end of the container for defining an enclosure forcontaining a biological fluid having multiple components; a floatassembly slideably disposed within the container and partitioning thecontainer into a lower volume zone and an upper volume zone; the floatassembly comprising: a lower cylindrical portion including an invertedhemispherically shaped interior surface surmounted by an interiorceiling surface for defining an inverted domed shaped chamber forisolating a target component of the biological fluid; an uppercylindrical portion surmounting the lower cylindrical portion andincluding a conically shaped upper surface defining a funnel shapedcavity converging towards the lower cylindrical portion of the float forreceiving, directing, and promoting biological fluid flow from the uppervolume zone toward the inverted domed shaped chamber; an open endedentrance passageway disposed within the float, for providing open fluidcommunication between the funnel shaped cavity and the inverted domedshaped chamber; an open ended exit passageway disposed within the floatfor providing open communication between the inverted domed shapedchamber and the lower volume zone; a first valve means for selectivelyopening and closing the open ended entrance passageway as a function ofa first pressure differential on the first valve means for controllingfluid flow from the funnel shaped cavity to the inverted dome shapedchamber and precluding fluid back flow through the first valve meansfrom the inverted dome shaped chamber to the funnel shaped cavity; asecond valve means for selectively opening and closing the open endedexit passageway as a function of a second pressure differential on thesecond valve means for controlling fluid flow out of the inverted domeshaped chamber to the lower volume zone of the container and precludingfluid back flow through the second valve means from the lower volumezone to the inverted domed shaped chamber, and wherein undercentrifugation the first valve means and the second valve meansinitially allow components of the biological fluid to flow through theinverted domed shaped chamber and subsequently close to seal theinverted domed shaped chamber as the function of the first pressuredifferential on the first valve means and as the function of the secondpressure differential on the second valve means for isolating the targetcomponent of the biological fluid within the inverted domed shapedchamber of the float.

Accordingly, it should be apparent that numerous modifications andadaptations may he resorted to without departing from the scope and fairmeaning of the claims as set forth hereinbelow following the detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an embodiment of a device for separatingand isolating components of a biological fluid.

FIG. 2 is a sectional view of an embodiment of a main centrifuge tube ofthe device.

FIG. 3 is an exploded parts view of an embodiment of a cap and valveassembly of the device.

FIG. 4 is a top plan view of the cap illustrated in FIG. 3.

FIG. 5 is a bottom plan view of the cap illustrated in FIG. 3.

FIG. 6 is a sectional view of an embodiment of a funnel float assemblyof the device illustrated in FIG. 1.

FIG. 7 is a sectional view of an embodiment of a float body of thefunnel float assembly illustrated in FIG. 6.

FIG. 8 is a top plan view of the float body illustrated in FIG. 7.

FIG. 9 is a bottom plan view of the float body illustrated in FIG. 7.

FIG. 10 is a perspective view of an embodiment of a funnel float cap ofthe funnel float assembly illustrated in FIG. 6.

FIG. 11 is a top plan view of the funnel float cap illustrated in FIG.10.

FIG. 12 is a bottom plan view of the funnel float cap illustrated inFIG. 10.

FIG. 13 is a sectional view of the funnel float cap illustrated in FIG.10.

FIG. 14 is a perspective view of an embodiment of an umbrella valve.

FIG. 15 is a perspective view of an embodiment of a duckbill valve.

FIG. 16 is a perspective view of the device being filled with abiological fluid.

FIG. 17 is a perspective view of the device after being filled with thebiological fluid and before centrifugation.

FIG. 18 is a perspective view of the device after a centrifugationprocess.

FIG. 19 is a perspective view of an isolated target component beingharvested from the device.

FIG. 20 is a block diagram of a conventional centrifuge and stirrerbeing employed with the device.

FIG. 21 is a block diagram of an embodiment of a method for utilizingthe device.

FIG. 22 is a perspective view of another embodiment of the device forseparating and isolating components of a biological fluid.

FIG. 23 is an exploded parts perspective view of the device illustratedin FIG. 22.

FIG. 24 is a sectional view of another embodiment of a main centrifugetube.

FIG. 25 is a sectional view of another embodiment of a funnel floatassembly.

FIG. 26 is an exploded parts view of the funnel float assemblyillustrated in FIG. 25.

FIG. 27 is a sectional view of an embodiment of a float body of thefunnel float assembly illustrated in FIG. 25.

FIG. 28 is a bottom perspective view of the float body illustrated inFIG. 27.

FIG. 29 is a top plan view of the float body illustrated in FIG. 27.

FIG. 30 is a perspective view of an embodiment of a funnel float cap ofthe funnel float assembly illustrated in FIG. 25.

FIG. 31 is a sectional view of the funnel float cap illustrated in FIG.30.

FIG. 32 is a top plan of the funnel float cap illustrated in FIG. 30.

FIG. 33 is a bottom plan view of the funnel float cap illustrated inFIG. 30.

FIG. 34 is a perspective view of a bottom cap of the funnel floatassembly illustrated in FIG. 25.

FIG. 35 is a sectional view of the bottom cap illustrated in FIG. 34.

FIG. 36 is a bottom plan view of the bottom cap illustrated in FIG. 33.

FIG. 37 is a perspective view of another embodiment of a bottom cap forthe funnel float assembly illustrated in FIG. 25.

FIG. 38 is a sectional view of the bottom cap illustrated in FIG. 37.

FIG. 39 is a bottom plan view of the bottom cap illustrated in FIG. 37.

FIG. 40 is an exploded parts perspective view of a plurality of densitytuning weights and the bottom cap illustrated in FIG. 37.

FIG. 41 is a perspective view of the coupling of the plurality ofdensity tuning weights with the bottom cap illustrated in FIG. 37.

FIG. 42 is a perspective view of the bottom cap illustrated in FIG. 34with a plurality of weight cradles added thereto.

FIG. 43 is a perspective view of the coupling of the plurality ofdensity tuning weights with the bottom cap illustrated in FIG. 42.

FIG. 44 is an exploded parts perspective view of a plurality of densitytuning weights and the float body of the funnel float assemblyillustrated in FIG. 6.

DETAILED DESCRIPTION OF THE INVENTION

Considering the drawings, wherein like reference numerals denote likeparts throughout the various drawing figures, reference numeral 10 isdirected to an embodiment of a device for separating and isolatingcomponents of a biological fluid and reference numeral 310 is directedto another embodiment of the device for separating and isolatingcomponents of a biological fluid.

Referring now to FIG. 1, and in one embodiment, the device 10 iscomprised of a main centrifuge tube or container 12, a tube cap assembly46 for selectively closing the container 12 for defining an enclosure 48for receiving and containing a biological fluid having multiplecomponents, a float assembly 130 partitioning the enclosure into a firstor lower volume zone 260 below the float assembly 130 and a second orupper volume zone 262 above the float assembly 130, and a flexible tube124 operatively coupled between the float assembly 130 and the tube capassembly 46 for traveling up or down with the float assembly 130 bycoiling or elongating respectively.

Main Centrifuge Tube 12

More specifically, and referring to FIGS. 1 and 2, the main centrifugetube or container 12 comprises a closed bottom end 14, a substantiallyflat annular top edge 16 defining an open top end 18, and a cylindricalsidewall 20 extending between the closed bottom end 14 and the annulartop edge 16. The cylindrical sidewall 20 includes an outer cylindricalsurface 22 and an inner circumferential or cylindrical surface 24 whichdefines a cylindrically shaped containing chamber 26 extending along acentral longitudinal axis 28 of the tube 12 which is also the centrallongitudinal axis of the device 10.

In one embodiment, the closed bottom end 14 is substantially formed as adisk shaped member having an interior surface 30 and an exterior surface32. The interior surface 30 includes an inner radiused edgetransitioning into the inner cylindrical surface 24 of the cylindricalsidewall 20. Similarly, the exterior face 32 includes an outer radiusededge transitioning into the outer cylindrical surface 22 of thecylindrical sidewall 20. Additionally, the closed bottom end 14 may beselectively closeable or integrally formed with the cylindrical sidewall20.

Furthermore, the tube is preferably constructed of but not limited to, amaterial which is both biocompatible and stable for gamma irradiation.In one embodiment, the tube 12 is constructed of, but not limited to, apolycarbonate or polystyrene material.

Standoff Member 34

Still referring to FIGS. 1 and 2, the tube 12 further comprises an openended hollow cylindrical standoff member 34 coaxially disposed withinthe main centrifuge tube 12 and comprised of a circular bottom edge 36coupled to or integrally formed with the closed bottom end 14, acircular top edge 38 defining an open top end 40, and a cylindricalsidewall 42 extending between the circular bottom edge 36 and thecircular top edge 38. The cylindrical sidewall 42 includes an interiorsurface 44 defining a cylindrically shaped receiving chamber 45concentrically disposed with the central longitudinal axis 28 of themain centrifuge tube 12. In one embodiment, the standoff member 34 hasan outer diameter and a height which are both substantially less than arespective outer diameter and height of the main centrifuge tube 12. Thefunction of the standoff member 34 will be further delineatedhereinbelow. Moreover, and in one embodiment, the standoff member 34 isconstructed of but not limited to, the same material as the maincentrifuge tube 12.

Tube Cap Assembly 48

Referring to FIG. 3, and in one embodiment, the device 10 is furthercomprised of the tube cap assembly 48 which comprises a tube cap 50, ahydrophobic air filter 80, an inlet valve 86, an outlet or extractionvalve 100, and a barb fitting 114.

Tube Cap 50

Referring now to FIGS. 3 through 5, the tube cap 50 is comprised of anupper annular portion 52 extending from an upper surface 54 of the tubecap 50 to a lower annular portion 56 having a lesser diameter than upperannular portion 52 and terminating to a lower surface 58 of the tube cap50. The upper annular portion 52 extends over and abuts annular top edge16 of cylindrical sidewall 20 while the lower annular portion 56 extendsinto and forms an interference fit with inner cylindrical surface 24 ofcylindrical sidewall 20 for providing a coupling between the tube cap 50and the main centrifuge tube 12 such that the tube cap 50 fits over andis maintained in place in open top end 18. In one embodiment, the tubecap 50 is medically bonded to the main centrifuge tube 12.

The tube cap 50 is also preferably constructed of, but not limited to, amaterial which is both biocompatibie and stable for gamma irradiation.In one embodiment, the tube cap 50 is constructed of, but not limitedto, a polycarbonate material.

Continuing to refer to FIGS. 3 through 5, the tub cap 50 is furthercomprised of a passageway housing 60 attached to or integrally formedwith the upper annular portion 52 of the tube cap 50. The passagewayhousing 60 upwardly extends from the upper surface 54 of the tube cap 50and includes an L-shaped passageway 62 having a first passageway branch64 extending between a passageway branch bend 66 and a housing inletport 68 recessed and disposed in the lower surface 58 of the tub cap 50.In turn, a second passageway branch 70 of the L-shaped extractionpassageway 62 extends between the passageway branch bend 66 and ahousing outlet port 72 disposed in a housing sidewall 74 of thepassageway housing 60. The second passageway branch 70 includes a taperextending away from the housing outlet port 72 and terminating prior oreaching branch bend 66.

The tub cap 50 is preferably constructed of, but not limited to, amaterial which is both biocompatible and stable for gamma irradiation,in one embodiment, the tub cap 50 is constructed of, but not limited to,a polycarbonate or polystyrene material.

Moreover, the tub cap 50 further includes an inlet valve aperture 76disposed through the tube cap 50 proximate one side of the passagewayhousing 60 and an air filter aperture 78 disposed through the tube cap50 proximate the other side of the passageway housing 60.

Air Filter 80, Inlet Valve 86, Extraction Valve 100, Barb Fitting 114

Still referring to FIGS. 3 through 5, the tube cap assembly 48 comprisesthe hydrophobic air filter 80 disposed in the air filter aperture 78 andcomprised of a flange 82 that abuts a recessed ledge 84 disposed inupper annular portion 52 of the tube cap 50 at a location circumscribingthe air filter aperture 78.

The hydrophobic air filter 80 is also preferably constructed of, but notlimited to, a material which is both biocompatible and stable for gammairradiation, in one embodiment, the hydrophobic air filter 80 isconstructed of, but not limited to, a polypropylene material with a PTFEfilter material. One example of the hydrophobic air filter 80 iscommercially available as part number X5009 from Qosina.

Additionally, the tube cap assembly 48 comprises the inlet valve 86which includes a conventional male luer lock head 88 having externalthreads 90. The head 88 transitions into a shoulder 92 of a cylindricalbody 94 which terminates to an underside 96 which, in turn, transitionsinto to a lower tapered end 98. The lower tapered end 98 of the inletvalve 86 is disposed through the inlet valve aperture 76 until theunderside 96 of inlet valve 86 abuts the upper surface 54 of the tubecap 50.

Furthermore, the tube cap assembly 48 comprises the outlet or extractionvalve 100 which, in one embodiment, includes a conventional male luerlock head 102 having external threads 104. The head 102 transitions intoa shoulder 106 of a cylindrical body 108 which terminates to anunderside 110 which, in turn, transitions into to a lower tapered end112. The lower tapered end 112 of the extraction valve 100 is disposedthrough the housing outlet port 72 until the underside 110 of theextraction valve 100 abuts the housing sidewall 74 of the passagewayhousing 60.

The inlet valve 86 and the outlet or extraction valve 100 are also bothpreferably constructed of, but not limited to, a material which is bothbiocompatible and stable for gamma irradiation. In one embodiment, boththe inlet valve 86 and the outlet or extraction valve 100 areconstructed of but not limited to, a polycarbonate material with asilicone rubber insert material. One example of the inlet valve 86 andthe outlet or extraction valve 100 is commercially available as partnumber 245501024 from Halkey-Roberts.

Moreover, the tube cap assembly comprises the barb fitting 114 which, inone embodiment, includes a short cylindrical portion 116 disposed in thehousing inlet port 68, a flange 118 transitioning from the shortcylindrical portion 116 and abutting a recessed ledge 120 disposed inlower annular portion 56 at a location circumscribing the housing inletport 68, and a barbed end 122 transitioning from the flange 118 andoperatively coupled to an upper end 126 of the coiled tube 124 as shownin FIG. 1. In turn, the coiled tube 124 includes a lower end 128 coupledto the float assembly 130 as will be further delineated hereinbelow.

The barb fitting 114 is also preferably constructed of, but not limitedto, a material which is both biocompatible and stable for gammairradiation. In one embodiment, the barb fitting 114 is constructed of,but not limited to, an ABS material. One example of the barb fitting 114is commercially available as part number BDMR210-81 from Value Plastics,Inc.

Float Assembly 130

Referring now to FIGS. 1 and 6, and as noted hereinabove, the device 10comprises float assembly 130 which can be defined as a dual densitysubsurface funnel and isolation float assembly 130 which is slideablydisposed within the container 12 and which partitions the cc tarn r 12into the first or lower volume zone 260 below the float assembly 130,the second or upper volume zone 262 above the float assembly 130, and anisolation or third volume zone defined by an isolation chamber 208within the float assembly 130 as further delineated hereinbelow.

In one embodiment, the float assembly 130 is comprised of: a funnel andisolation float 132 comprised of a float body 134 and a float cap 194; afirst check valve means in the form of an umbrella valve 234; and asecond check valve means in the form of a duckbill valve 248. The funnelfloat 132 can be manufactured as multiple elements or as a single,unitary element.

Float Body 134

More specifically, and referring to FIGS. 6 and 7, the float body 134comprises a circular bottom surface 136, a frustum shaped upper topsurface 138, and a float sidewall 140 extending between the circularbottom surface 136 and the frustum shaped upper top surface 138. Thecircular bottom surface 136 can be substantially flat or taper toward acentral longitudinal axis 168 of the float body 134.

The float sidewall 140 includes an outer circumferential or cylindricalsurface 142 extending between a circular outer periphery 144 of thecircular bottom surface 136 and a circular outer edge 146 of the upperfrustum shaped top surface 138. The outer circumferential surface 142 ofthe float sidewall 140 defines a diameter that is less than a diameterdefined by the inner circumferential surface 24 of the main centrifugetube 12 for defining a circumferential gap 148 between the outercircumferential surface 142 of the float sidewall 140 and the innercircumferential surface 24 of the tube 12.

Additionally, the float sidewall 140 includes an inner hemisphericalsurface 150 defining an inverted domed shaped or hemispherical shapedcavity 152 extending from a radiused bottom section 154 of thehemispherical surface 150 to an upper annular ledge 156 defining andannular opening 158 of the inverted domed shaped cavity 152. The upperannular ledge 156 transitions into an inner cylindrical surface 160defining an open ended cylindrically shaped cavity 162 surmounting theinverted domed shaped cavity 152 such that the annular opening 158 alsodefines a lower opening of the cylindrically shaped cavity 162. In turn,the inner cylindrical surface 160 extends from the annular ledge 156 toa circular inner edge 164 of the frustum shaped upper top surface 138.The circular inner edge 164 circumscribes and defines an annular opening166 between the cylindrically shaped cavity 162 and the frustum shapedupper top surface 138 such that the opening 166 defines an upper openingof the cylindrically shaped cavity 162 and a lower opening of thefrustum shaped upper top surface 138.

Furthermore, the float body 134 is preferably constructed of, but notlimited to, a material which is both biocompatible and stable for gammairradiation. In one embodiment, the float body 134 is constructed of,but not limited to, a polystyrene or polycarbonate type of material.

Exit Passageway 170 and Extraction Passageway 180

Referring to FIGS. 7 through 9, the float body 134 further comprises anexit passageway 170 extending between an first circular exit port 172disposed in the radiused bottom section 154 of the inner hemisphericalsurface 150 of the float body 134 and a second circular exit port 174disposed in the bottom surface 136 of the float body 134 for providingopen fluid communication between the inverted domed shaped cavity 152and the first or lower volume zone 260 below the float assembly 130.Additionally, and in one embodiment, the first and second circular exitports have a common axis defined by the central longitudinal axis 168 ofthe float body 134. Furthermore, the second circular exit port 174 iscircumscribed by an annular shoulder 176 which steps down and into anannular recessed area 178 disposed through the bottom surface 136 of thefloat body 134.

Still referring to FIGS. 7 through 9, the float body 134 furthercomprises an L-shaped extraction passageway 180 defined by a firstbranch 182, a second branch 184, and a branch bend 186 therebetween. Thefirst branch 182 is formed by providing a bore through both the outercircumferential surface 142 and the float sidewall 140 at an anglesubstantially perpendicular to the central longitudinal axis 168 of thefunnel float body 134 wherein the outer circumferential surface 142 isplugged thereafter with plug 145. The second branch 184 is formed byproviding a bore through both the frustum shaped upper top surface 138and the float sidewall 140 which terminates in the first branch 182 atbend 186 and which is at an angle substantially parallel to the centrallongitudinal axis 168 of the funnel float body 134. The L-shapedextraction passageway 180 is in open fluid communication with andextends between a tear drop shaped port 188 disposed in the innerhemispherical surface 150 and a circular shaped port 190 disposed in thefrustum shaped upper top surface 138. An upper portion 192 of the secondbranch 184 has an increased diameter proximate the circular shaped port190 for receiving the lower end 128 of the coiled tube 124 which has itsupper end 126 coupled to the barbed end 122 of the barb fitting 114 forproviding open fluid communication between the inverted domed shapedcavity 152 and the extraction valve 100.

Float Cap 194

Referring now to FIGS. 10 through 13, the funnel and isolation float 132is further comprised of the float cap 194 which comprises a circularbottom surface 196, a frustum shaped lower top surface 198, and a floatcap circumferential sidewall 200 extending between the circular bottomsurface 196 and the frustum shaped lower top surface 198.

The circumferential sidewall 200 includes an outer circumferential orcylindrical surface 202 which extends between a circular outer periphery204 of the circular bottom surface 196 and a circular outer edge 206 ofthe frustum shaped lower top surface 198.

The float cap 194 is complementally shaped and sized to fit within thecylindrically shaped cavity 162 of the float body 134 for closing theannular opening 158 of the inverted domed shaped cavity 152 for definingan inverted dome shaped isolation chamber 208 as illustrated in FIG. 6.

Additionally, and referring to FIGS. 6 and 10, the frustum shaped lowertop surface 198 of the float cap 194 provides a lower continuation ofthe frustum shaped upper top surface 138 of the float body 134 fordefining a conical or funnel shaped surface 210 of the funnel andisolation float 132 which, in turn, defines a conical or funnel shapedcavity 212. In one embodiment, the frustum shaped upper top surface 138and lower top surface 198 are continuous with one another such that thefunnel shaped surface 210 uniformly tapers from the circular outer edge146 of the frustum shaped upper top surface 138 to a lower annular edge214 of the frustum shaped lower top surface 198. In another embodiment,the lower top surface 198 may have an accelerated tapering.

The funnel shaped surface 210 inwardly tapers from the circular outeredge 146 of the frustum shaped upper top surface 138 to the lowerannular edge 214 of the frustum shaped lower top surface 198 where thefunnel shaped surface 210 transitions into a funnel tube portion 216 ofthe float cap 194. The funnel tube portion 216 defines a central openended cylindrical opening 218 which extends through a central area ofthe float cap 194. In turn, at least one funnel fluid passageway 220 isdisposed through the funnel float cap 194 between a funnel port 222(FIG. 11) disposed in the frustum shaped lower top surface 198 and anentrance port 224 (FIG. 12) disposed in the bottom surface 196 of thefloat cap at location adjacent the funnel tube portion 216 therebyproviding open communication between the funnel shaped cavity 212 andthe inverted dome shaped isolation chamber 208. In one embodiment, thereare four funnel fluid passageways 220 connected between respective ports222, 224 and equally spaced apart at ninety degree intervals aroundfunnel tube portion 216.

Additionally, and in one embodiment, the funnel fluid passageways 220are generally triangular in shape with a concave apex having roundededges located proximate the funnel tube portion 216 and a convex basehaving rounded edges located distal from the funnel tube portion 216 asillustrated in FIG. 11. Furthermore, and in one embodiment, the funnelshaped surface 210 has a preferred declivity of about thirty degreesfrom a plane perpendicular to a central axis 226 (FIG. 13) of the floatcap 194. Thus, this provides the funnel face with about a one hundredtwenty degree opening.

Furthermore, and referring to FIGS. 12 and. 13, one embodiment of thecircular bottom surface 196 of float cap 194 is comprised of: asubstantially flat surface 228 circumscribing the central open endedcylindrical opening 218 and the plurality of funnel fluid passageways220, an annular portion 230 transitioning from the surface 228 andhaving an declivity of about five degrees from a plane perpendicular tothe central axis 226 of the funnel cap 134, and a substantially flatannular bottom edge surface 232 transitioning from annular portion 230.

Moreover, the float cap 194 is preferably constructed of, but notlimited to, a material which is both biocompatible and stable for gammairradiation. In one embodiment, the float cap 194 is constructed of, butnot limited to, a polystyrene type of material.

Umbrella Valve 234

Referring to FIGS. 6 and 14, and one embodiment, the float assembly 130further comprises a first one way valve or check valve in the form of anelastic umbrella valve 234 which is used to selectively open and closethe funnel fluid passageway's 220 based on a pressure differential ofbiological fluid on the umbrella valve 234 thereby controlling fluidflow from the funnel shaped cavity 212 to the inverted dome shapedisolation chamber 208 while precluding fluid from flowing back out ofthe inverted dome shaped isolation chamber 208 to the funnel Shapedcavity 212 via the umbrella valve 234. Thus, the umbrella valve 234provides a unidirectional flow of biological fluid from the second orupper volume zone 262 above the float to the third volume zone orisolation zone defined by inverted dome shaped isolation chamber 208 asa function of the pressure differential of biological fluid on theumbrella valve 234.

More specifically, and in one embodiment, the umbrella valve 234 iscomprised of a generally circular canopy or dome 236 which, in anunstressed position, extends generally perpendicularly to a centrallylocated stem 238. The stem 238 includes a bulbous portion 240 beyondwhich is a tapered shaft portion 242. In one embodiment, the entireumbrella valve 234 is a one piece, integral construction.

Referring to FIGS. 6, 10, 12, and 14, the umbrella valve 234 is mountedto the float cap 194 by securing the stem 238 through the central openended cylindrical opening 218 disposed through the float cap 194. Thisis accomplished by sizing the length of stern 238 between the canopy 236and the bulbous portion 240 greater than the length of the cylindricalopening 218 and sizing the diameter of the bulbous portion greater thanthe diameter of the cylindrical opening 218 such that when the taperedshaft portion 242 of the stem 238 is inserted into the cylindricalopening 218 from the bottom surface 196 of the float cap 194 and pulledor stretched away from the frustum shaped lower top surface 198 of thefloat cap 194 the elastic bulbous portion 240 passes through the centralopen ended cylindrical opening 218 and resumes its normal shape adjacentthe frustum shaped lower top surface 198 of the float cap 194 therebyacting as an anchor for holding the umbrella valve 234 in place. Afteranchoring the umbrella valve 234 in place, the tapered shaft portion 242may be trimmed while retaining the bulbous portion 240.

Additionally, the generally circular canopy or dome 236 includes a flatunderside contact surface 244 when in the unstressed position. Thus,when the centrally located stem 238 is stretched an axial force isexerted on the canopy or dome 236 such that the underside 244 is drawninto a normal tight, sealing contact against the substantially flatsurface 228 of the bottom surface 196 of the float cap 194 for sealingthe funnel fluid passageways 220.

With this construction, the elastic umbrella valve 234 provides a oneway valve which controls fluid flow into the inverted dome shapedisolation chamber 208 and precludes fluid flow out of the inverted domeshaped isolation chamber 208 via the elastic umbrella valve 234. Inparticular, the elastic umbrella valve 234 opens under a predeterminedpressure differential or cracking pressure or, in other words, when thepressure in the funnel fluid passageways 220 is greater than below or onan outer surface 246 of the canopy or dome 236 by a predetermined orknown cracking pressure of the umbrella valve 234. Hence, when there isa positive pressure differential defined by a pressure at the funnelfluid passageways 220 being greater than the pressure on the outersurface 246 of the canopy or dome 236 by an amount which is greater thanthe cracking pressure then the pressure differential causes the flexiblecanopy or dome 236 to invert or flex downwardly, away from the bottomsurface 196 of the float cap 194 thereby permitting the biological fluidto pass into the inverted dome shaped isolation chamber 208. After thepressure differential resides to a point less than the cracking oropening pressure, the resilient canopy or dome 236 resumes its sealingposition under the funnel fluid passageways 220 thereby precluding anyfurther biological fluid from entering the inverted dome shapedisolation chamber 208.

In one embodiment, the umbrella valve 234 is preferably constructed of,but not limited to, a material which is both biocompatible and stablefor gamma irradiation. In one embodiment, the umbrella valve 234 isconstructed of but not limited to, a silicone type of material; however,any sufficiently flexible and resilient material may be employed as longas the material is compatible with the biological fluid being processedand preferably, biocompatible and stable for gamma irradiation. Oneexample of the umbrella valve 234 is commercially available as partnumber 2510-102 from Vernay.

Duckbill Valve 248

Still referring to FIGS. 6, 7, and 15, and one embodiment, the floatassembly 130 further comprises a second one way valve or check valve inthe form of an elastic duckbill valve 248 which is used to selectivelyopen and close the exit passageway 170 extending between the firstcircular exit port 172 and the second circular exit port 174 based on apressure differential of biological fluid on the duckbill valve 248thereby controlling fluid flow from the inverted dome shaped isolationchamber 208 to the first or lower volume zone 260 while precluding fluidfrom flowing back into the inverted dome shaped isolation chamber 208from the first or lower volume zone 260 via the duckbill valve 248.Thus, the duckbill valve 248 provides a unidirectional flow ofbiological fluid from the isolation zone within the float to the firstor lower volume zone 260 as a function of the pressure differential ofbiological fluid on the duckbill valve 248.

In one embodiment, the duckbill valve 248 is comprised of an open endedhollow cylindrical portion 250 coupled to the exit passageway 170 andtransitioning from a radially outwardly projecting annular flangeportion 252 to a hollow V-shaped or converging portion 254 whichterminates to an elongated outlet slit 256 defined by a pair ofresilient sealing lips 258. The resilient sealing lips 258 are formed tomove apart to open the slit 256 for fluid to flow in one directionthrough a fluid passageway axially extending through the duckbill valve248.

The resilient sealing lips 258 normally maintain the slit 256 in theclosed position. When fluid pressure within the hollow V-shaped orconverging portion at a location above the sealing lips 258 is greaterthan a fluid pressure below the resilient sealing lips 258 by apredefined cracking pressure of the duckbill valve 248 then theresilient sealing lips 258 spreading and the slit 256 open therebypermitting fluid to flow downwardly through the exit passageway oraperture 170 and through the axially extending fluid passageway of theduckbill valve 248. The outlet slit 256 closes when the fluid pressurebelow or on the outer surfaces of the resilient sealing lips 258 ishigher than the fluid pressure above the resilient sealing lips 258 bymore than the cracking pressure of the duckbill valve 248 therebypreventing the backflow of fluid through the duckbill valve.Additionally, if fluid flow stops or reverses direction, theback-pressure exerted by the fluid upon the outer surfaces of theresilient sealing lips 258 forces the lips into sealing engagementagainst one another, closing outlet slit 256 and preventing fluidbackflow.

As assembled, the annular flange portion 252 of the duckbill valve isseated within the annular recessed area 178 disposed through the bottomsurface 136 of the float body 134 while the hollow cylindrical portion250 fits over the annular shoulder 176 such that the exit passageway 170is in open communication with the axially extending fluid passageway ofthe duckbill valve 248 and such that a press type fit or coupling isprovide between the duckbill valve 248 and annular shoulder 176 of thefloat body 134 to maintain a seal between the duckbill valve 248 and thefloat body 134.

In one embodiment, the duckbill valve 248 is preferably constructed of,but not limited to, a material which is both biocompatible and stablefor gamma irradiation and is constructed of but not limited to, asilicone type of material; however, any sufficiently flexible andresilient material may be employed as long as the material is compatiblewith the biological fluid being processed and preferably, biodegradableand stable for gamma irradiation. One example of this embodiment of theduckbill valve is commercially available as part number DU054.001-154.01 from Mini Valve International.

Use and Operation

In use and operation, and referring to the drawings, the device 10initially receives a multiple component biological fluid sample (e.g.,peripheral blood, bone marrow aspirate, blood components, cord blood,apheresis blood products, lipo-aspirate, semen, urine, milk, ascitesfluid, exudate or cerebrospinal fluids) by performing the steps ofremoving the cap 85 from the male luer lock head 88 of the inlet valve86, coupling a conventional needleless syringe 270 or other dispensingdevice containing the biological fluid sample to the male luer lock head88 of the inlet valve 86, injecting or dispensing the biological fluidsample into the device 10 from the conventional syringe 270 or otherdispensing device (FIG. 16), decoupling the conventional syringe 270 orother dispensing device from the male luer lock head 88 of the inletvalve 86, and reattaching cap 85. When supplying the device 10 withbiological fluid, the air filter allows air to escape from the enclosure48.

The received biological fluid sample fills the second or upper volumezone 262 above the inverted dome shaped isolation chamber 208 includingthe funnel shaped cavity 212 and flows to the first or lower volume zone260 below the float assembly 130 via the circumferential gap 148disposed between the outer circumferential surface 142 of the floatsidewall 140 and the inner circumferential surface 24 of the tube 12 asillustrated in FIG. 17. The biological fluid sample is initiallyprecluded from entering or passing through the inverted dome shapedisolation chamber 208 by the normally closed umbrella valve 234 and thenormally closed duckbill valve 248. Additionally, the circular top edge38 of the standoff member 34 abuts the circular bottom surface 136 ofthe float body 134 of the float assembly 130 for providing an initiallift off the float assembly 130 from the interior surface 30 of theclosed bottom end 14 of the tube 12 for precluding a vacuum seal fromforming therebetween while also protecting the elastic duckbill valve248 in the cylindrically shaped receiving chamber 46 of the standoffmember 34 under initial conditions.

Once the biological fluid sample has been received within the enclosure48 defined by the tube 12 and tube cap 50, the device 10 is placed in aconventional centrifugation device 280 which is operated for one or morepredetermined durations at one or more predetermined speeds. In oneembodiment, and for a biological fluid sample exemplified by blood, theconventional centrifugation device 280 is operated for about 12 to about15 minutes at about 3,200 RPM. Of course, one or more time durations andone or more speeds can be empirically determined for a specificbiological fluid to be processed by the device 10 and may vary from onebiological fluid to another.

Upon initial centrifugation of the device 10, the biological fluid inthe funnel shaped cavity 212 increasingly applies a pressure on theumbrella valve 234 which results in a pressure differential on theumbrella valve 234 becoming greater than the cracking pressure of theumbrella valve 234 thereby allowing fluid to flow through one or more ofthe funnel fluid passageways 220 and into the inverted dome shapedisolation chamber 208 which begins to fill and apply a second pressureon the duckbill valve 248 which results in a pressure differential onthe duckbill valve 248 being greater than the cracking pressure of theduckbill valve 248 thereby allowing biological fluid to initially flowout of the inverted dome shaped isolation chamber 208 via exitpassageway 170. At first, the biological fluid quickly flows through theinverted dome shaped isolation chamber 208 and when the sample is bloodthe hematocrit is initially not concentrated. With both the umbrellavalve 234 and duckbill valve 248 in an open position, the building ofthe column of biological fluid in the first or lower volume zone 260 orbelow the float assembly 130 continues. As this column of the biologicalfluid builds, the device 10 provides a unique circulation process ordensity feedback process which will he clearly delineated by usingperipheral blood as an example of the biological fluid being processed.

Accordingly, and as the biological fluid such as peripheral bloodstratify based on density and the components build the column, the cellsthat are just below the bottom surface 136 of the float body 134 alsocontinue to stratify such that the cell density above the level of theduckbill valve 248 is less than the cell density below the level of theduckbill valve 248 by an amount which provides a pressure differentialwhich is less than the cracking pressure of the duckbill valve 248thereby resulting in the closure of the duckbill valve 248 such thatover time the reds cells accumulate at the bottom section 154 of theinverted dome shaped isolation chamber 208 and become packed and thus,there are packed cells at the bottom section 154 of the chamber whichhave a greater density than the cells right below the level of theduckbill valve 248 because at the same time that the density of redcells is increasing at the bottom section 154 the density right belowthe duckbill valve 248 is decreasing because greater density cells arealso migrating toward the bottom end 14 of the tube 12. Thus, as thisdensity increases a pressure differential builds to surpass the crackingpressure of the duckbill valve 248 which then opens thereby allowing thered cells to flow through the exit passageway 170 and displace thebiological fluid in the first or lower zone 260 which, in turn, pushesfluid up or causes a surge of fluid up through the circumferential gap148 between the outer circumferential surface 142 of the float sidewall140 and the inner circumferential surface 24 of the tube 12. This upwardfluid flow has the effect of carrying the lighter components of thebiological fluid including the white blood cells and platelets from thefirst or lower volume zone 260 of the tube 12 back up to the second orupper volume zone 262 of the tube 12 wherein these lighter componentsare heavier than the plasma in the second or upper volume zone 262 thatthey got circulated into so the white blood cells and platelets or buffycoat included therein fall back down through the funnel shaped cavity212, past the umbrella valve 234, and collect in the inverted domeshaped isolation chamber 208. This cell circulation process or densityfeedback process continues on towards an equilibrium where the pressuredifferential or the density differential of the cells above and belowduckbill valve 248 is less than the cracking pressure of the duckbillvalve 248 resulting in the duckbill valve 248 having a final closure andsimilarly the density differential of the cells above and below umbrellavalve 234 is less than the cracking pressure of the umbrella valve 234resulting in the umbrella valve 234 having a final closure therebyconcluding the unique cell circulation process or density feedbackprocess of the device 10.

Hence, this cell circulation process or density feedback process allowsmultiple chances at capturing the white cells or other target componentsof interest in the inverted dome shaped isolation chamber 208 therebyproviding higher recovery rates as compared to know prior art devices.

Following the centrifugation step, the device 10 containing theseparated and isolated biological fluid components is removed from thecentrifugation device 280 (FIG. 18) and placed on a conventionalmagnetic stirrer 290 as diagrammatically shown in FIG. 20. The stirreris then energized for activating or coacting with a magnetic stirringbar 292 located inside the inverted dome shaped isolation chamber 208for stirring at least one isolated target component within the isolationchamber 208 for increasing a recovery rate of a subsequent harvestingstep. In one embodiment, at least the one isolated target component orfraction is stirred for about twenty seconds. Of course, stirring timesmay vary from one biological fluid to another and a specific stirringtime for a specific biological fluid can be empirically determined.

Following the stirring step, the harvesting step of at least the oneisolated target component or fraction is performed and is comprised ofthe steps of removing the cap 99 from the male luer lock head 102 of theextraction valve 100, coupling a conventional syringe 272 or otherextraction device to the male luer lock head 102 of the extraction valve100, aspirating at least the one isolated target component or fractionfrom the inverted dome shaped isolation chamber 208 by providing avacuum from the syringe 103 to the tear drop shaped port 188 disposed inthe radiused bottom section 154 of the inner hemispherical surface 150and aspirating at least the one isolated target component or fractionfrom the inverted dome shaped isolation chamber 208 and through the teardrop shaped port 188 and into and through the exit L-shaped extractionpassageway 180 disposed in the float body 134, the coiled tube 124, thebarb fitting 114, the L-shaped housing passageway 62, the extractionvalve 100, and finally into the conventional syringe 272 (FIG. 19).

After the harvesting step is completed, the conventional syringe 103 isdecoupled from the male luer lock head 102 of the extraction valve 100in preparation for at least one diagnostic or therapeutic application ofat least the one target component or fraction. When harvesting at leastthe one target component or fraction, the air filter allows air into theenclosure 48. Additionally, when the cracking pressure of the umbrellavalve is reached during a harvesting step, plasma flows into theinverted dome shaped isolation chamber 208 to displace the removedvolume of at least the one target component or fraction.

It should be noted that a counter weight device 282 may be used in theconventional centrifugation device 280 to offset the weight of thedevice 10 containing biological fluid as required.

Furthermore, device 10 should be assembled in an environment to minimizethe risk of particulate matter in the fluid path and when used forclinical applications, the device 10 must be sterilized and the fluidpath should be non-pyrogenic.

Device 310

Referring now to FIGS. 22 and 23, and in one embodiment, the device 310is comprised of a main centrifuge tube or container 312; the tube capassembly 46 (also detailed in FIGS. 3 through 5) for selectively closingthe container 312 for defining an enclosure 334 for receiving andcontaining a biological fluid having multiple components; a dual densitysubsurface funnel and isolation float assembly 340 which is slideablydisposed within the container 312 and which partitions the enclosure 334into a first or lower volume zone 336 below the float assembly 340, asecond or upper volume zone 338 above the float assembly 340, and anisolation or third volume zone defined by an isolation chamber 378within the float assembly 340 as further delineated hereinbelow; and theflexible tube 124 (also detailed in FIG. 1) operatively coupled betweenthe float assembly 340 and the tube cap assembly 46 for traveling up ordown with the float assembly 340 by coiling or elongating respectively.

Main Centrifuge Tube 312

More specifically, and referring to FIGS. 24, the device 310 iscomprised of the main centrifuge tube or container 312 which comprises aclosed substantially flat annular bottom end 314, a substantially flatannular top edge 316 defining an open top end 318, and a cylindricalsidewall 320 extending between the closed bottom end 314 and the annulartop edge 316. The cylindrical sidewall 320 includes an outer cylindricalsurface 322 and an inner circumferential or cylindrical surface 324which defines a cylindrically shaped containing chamber 326 extendingalong a central longitudinal axis 328 of the tube 312 which is also thecentral longitudinal axis of the device 310.

In one embodiment, the closed bottom end 314 is substantially formed asa disk shaped member having an interior surface 330 and an exteriorsurface 332. The interior surface 330 includes an inner radiused edgetransitioning into the inner cylindrical surface 324 of the cylindricalsidewall 320. Similarly, the exterior face 332 includes an outerradiused edge transitioning into the outer cylindrical surface 322 ofthe cylindrical sidewall 320.

Additionally, the closed bottom end 314 may be selectively closeable orintegrally formed with the cylindrical sidewall 320.

Furthermore, the tube is preferably constructed of, but not limited to,a material which is both biocompatible and stable for gamma irradiation.In one embodiment, the tube 312 is constructed of but not limited to, apolycarbonate or polystyrene material.

Tube Cap Assembly 48

Referring to FIGS. 22 and 23, and back to FIGS. 3 through 5, the device310 is further comprised of the tube cap assembly 48 which is asdelineated in detail hereinabove and which will not be repeated so as tonot burden the record.

Float Assembly 340

Referring now to FIGS. 25 and 26, and as noted hereinabove, the device310 is further comprised of the float assembly 340 which comprises afunnel and isolation float 342 comprised of a float body 344, a funnelfloat cap 420, and a bottom float cap 480 or 500. Because the funnel andisolation float 342 should be made with precision, it is preferable tomake the funnel and isolation float 342 in subcomponents comprised ofthe float, body 344, funnel float cap 420, and the bottom float cap 480or 500 to facilitate precision injection molding and it is alsopreferable that the subcomponents be assembled using sonic weldingdevices or other reliable means.

The float assembly 340 further comprises the first check valve meanswhich, in one embodiment, is in the form of the umbrella valve 234detailed in FIG. 14; and the second check valve means which, in oneembodiment, is in the form of the duckbill valve 248 detailed in FIG.15.

Float Body 342

More specifically, and referring to FIGS. 25 through 27, the float body344 comprises an annular bottom edge 346, a frustum shaped top surface348, and a float body sidewall 350 extending between the annular bottomedge 346 and the frustum shaped upper top surface 350.

The float body sidewall 350 includes an outer circumferential orcylindrical surface 352 extending between the annular bottom edge 346and a circular outer periphery 354 of the frustum shaped top surface348. The outer circumferential surface 352 of the float body sidewall350 defines a diameter that is less than a diameter defined by the innercircumferential surface 324 of the main centrifuge tube 312 for defininga circumferential gap 356 between the outer circumferential surface 352of the float body sidewall 350 and the inner circumferential surface 324of the tube 312.

Additionally, the float body sidewall 350 includes an innerhemispherical surface 358 defining an inverted domed shaped orhemispherical shaped cavity 360 extending from a radiused bottom section362 of the hemispherical surface 358 to an upper annular ledge 364defining an annular opening 366 of the inverted domed shaped cavity 360.The upper annular ledge 364 transitions into an inner cylindricalsurface 368 defining an open ended cylindrically shaped cavity 370surmounting the inverted domed shaped cavity 360 such that the annularopening 366 also defines a lower opening of the cylindrically shapedcavity 370. In turn, the inner cylindrical surface 368 extends from theannular ledge 364 to a circular inner edge 372 of the frustum shaped topsurface 348. The circular inner edge 372 circumscribes and defines anannular opening 374 between the cylindrically shaped cavity 370 and thefrustum shaped top surface 348 such that the opening 374 defines anupper opening of the cylindrically shaped cavity 370 and a lower openingof the frustum shaped top surface 348.

Float body 344 is preferably constructed of, but not limited to, amaterial which is both biocompatible and stable for gamma irradiation.In one embodiment, the float body 344 is constructed of but not limitedto, a polystyrene or polycarbonate type of material,

Exit Aperture 380

Referring to FIGS. 27 and 28, the float body 344 further comprises anexit aperture or passageway 380 extending between a first circular exitport 382 disposed in the radiused bottom section 362 of the innerhemispherical surface 358 of the float body 344 and a second circularexit port 384 disposed in a bottom surface 386 of the float body 344 forproviding open fluid communication between the inverted domed shapedcavity 360 and the first or lower volume zone 336 below the floatassembly 340.

Additionally, the first and second circular exit ports 382, 384 have acommon axis defined by a central longitudinal axis 388 of the float body344.

Furthermore, the second circular exit port 384 is disposed in the bottomsurface 386 of the float body 344 at a location which is recessed fromthe annular bottom edge 346 of the float body sidewall 350 and whichdefines an intersection of two diametrically extending and intersectingcross members 390, 392 defining four spaced apart cavities 394, 396,398, and 400 in the bottom surface 386 of the float body 344. The fourspaced apart cavities 394, 396, 398, and 400 reveal a lower outersurface 402 of the inverted domed shaped cavity 360.

Extraction Aperture 404

Referring now to FIGS. 27 and 29, the float body 344 further comprisesan extraction aperture 404 which, in one embodiment, is an L-shapedextraction passageway 404 defined by a first branch 406, a second branch408, and a branch bend 410 therebetween. The first branch 406 is formedby providing a bore through the float body sidewall 350 at an anglesubstantially perpendicular to the central longitudinal axis 388 of thefloat body 344 Wherein a portion of the float body sidewall 350 adjacentthe outer circumferential surface 352 is plugged thereafter with plug412 as illustrated in FIG. 25. The second branch 408 is formed byproviding a bore through both the frustum shaped top surface 348 and thefloat body sidewall 350 which terminates in the first branch 406 at bend410 and which is at an angle substantially parallel to the centrallongitudinal axis 388 of the float body 344. The extraction aperture orL-shaped extraction passageway 404 is in open fluid communication withand extends between and an oval shaped port 414 disposed in the innerhemispherical surface 358 and a circular shaped port 416 disposed in thefrustum shaped top surface 348. An upper portion 418 of the secondbranch 408 has an increased diameter proximate the circular shaped port416 for receiving the lower end 128 of the coiled tube 124 which has itsupper end 126 coupled to the barbed end 122 of the barb fitting 114 forproviding open fluid communication between the inverted domed shapedcavity 360 and the extraction valve 100.

Funnel Float Cap 420

Referring now to FIGS. 30 through 33, the funnel and isolation float 342is further comprised of the funnel float cap 420 which comprises afrustoconical or funnel shaped wall 422 transitioning into and mountinga cylindrically shaped neck 460 transitioning into and mounting a diskedshaped base 470. The funnel shaped wall 422 includes a frustoconical orfunnel shaped upper surface 424 which defines a conical or funnel shapedcavity 426. The funnel shaped wall 422 further includes a frustoconicalor funnel shaped lower surface 428 and an outer circumferential orcylindrically shaped peripheral edge 430 extending between thefrustoconical or funnel shaped upper and lower surfaces 424,428.

The disked shaped base 470 is complementally shaped and sized to fitwithin the cylindrically shaped cavity 370 of the float body 344 andwhen fitted therein, an elastomeric 0-ring 472 is captured between alower peripheral chamfer 474 of the disk shaped base 470 and the annularledge 364 for providing a seal therebetween and for closing the annularopening 366 of the inverted domed shaped cavity 360 for defining aninverted dome shaped isolation chamber 378 as illustrated in FIG. 25.Additionally, the funnel shaped lower surface 428 of the funnel shapedwall 422 is complementally shaped and sized to abut against the frustumshaped top surface 348 of the float body 344 when the elastomeric o-ring472 is sealed against the annular ledge 364.

When the disked shaped base 470 is properly fitted within thecylindrically shaped cavity 370 of the float body 344 as delineatedabove, an alignment and interlocking means is provided between thefunnel float cap 420 and the float body 344. Specifically, and referringto FIGS. 25, 27, and 31, a circular segment 432 protruding from thefrustum shaped top surface 348 of the float body 344 is received withina circular segment indentation 434 disposed through the funnel shapedlower surface 428 and into the funnel shaped wall 422 such that theprotruding circular segment 432 aligns and interlocks with the circularsegment indentation 434. Additionally, the frustum shaped top surface348 of the float body 344 includes an annular projection 436 which has atriangularly shaped cross sectional area sized to be received within acomplementally shaped annular recess 438 (FIG. 25) disposed through thefunnel shaped lower surface 428 and into the funnel shaped wall 422 ofthe funnel float cap 420. Furthermore, the alignment and interlockingmeans also aligns an opening 440 extending through the funnel shapedwall 422 with the circular shaped port 416 of the extraction aperture orL-shaped extraction passageway 404 such that a portion of the coiledtube 124 proximate the lower end 128 is received therethrough.

Moreover, and referring to FIGS. 30 through 33, the frustoconical orfunnel shaped upper surface 424 of the funnel shaped wall 422 inwardlytapers from an upper annular outer edge 442 to a lower annular edge 444where the funnel shaped upper surface 424 transitions into a funnel tubeportion 446 of the funnel float cap 420. The funnel tube portion 446defines a central open ended cylindrical opening 448 which extendsthrough a central area of the cylindrically shaped neck 460 and thedisked shaped base 470 of the funnel float cap 420. In turn, at leastone funnel fluid passageway or aperture 450 is disposed through thefunnel float cap 420 at location adjacent the funnel tube portion 446thereby providing open communication between the funnel shaped cavity426 (FIG. 25) and the inverted dome shaped isolation chamber 378. In oneembodiment, there are four funnel fluid passageways 450 which areequally spaced apart at ninety degree intervals around funnel tubeportion 446.

Additionally, and in one embodiment, the funnel fluid passageways orapertures 450 are generally triangular in shape with a concave apexhaving rounded edges located proximate the funnel tube portion 446 and aconvex base having rounded edges located distal from the funnel tubeportion 446 as illustrated in FIGS. 30, 32, and 33. Furthermore, and inone embodiment, the funnel shaped upper surface 210 has a declivity ofabout thirty degrees from a plane perpendicular to a central axis 452(FIG. 31) of the float cap 420. Thus, this provides the funnel face withabout a one hundred twenty degree opening.

Furthermore, and referring to FIGS. 31 and 33, the lower peripheralchamfer 474 transitions into an annular recessed bottom area 476 which,into turn, transitions into substantially flat bottom surface 478circumscribing the central open ended cylindrical opening 448 and theplurality of funnel fluid passageways or apertures 450 as illustrated inFIG. 33.

Moreover, the funnel float cap 420 is preferably constructed of, but notlimited to, a material which is both biocompatible and stable for gammairradiation. In one embodiment, the funnel float cap 420 is constructedof, but not limited to, a polystyrene or polycarbonate type of material.

Bottom Cap 480

Referring now to FIGS. 34 through 36, the funnel and isolation float isfurther comprised of the bottom float cap 480 which comprises a circularwall 482 comprised of a substantially flat top surface 484, asubstantially flat bottom surface 486, and an outer peripheral edge 488extending therebetween. Additionally, the bottom float cap 480 comprisesan annular lip or projection 490 which protrudes from the substantiallyflat top surface 484 at a location adjacent the outer peripheral edge488 of the circular wall 482 and which has a triangularly shaped crosssectional area sized to be received within a complementally shapedannular recess 538 (FIG. 25) disposed in the float body 344.Furthermore, the bottom float cap 480 comprises a centrally locatedannular ledge 492 recessed below the substantially flat top surface 484of the circular wall 482. The centrally located annular ledge 492circumscribes a centrally located aperture 494 disposed through thecircular wall 482. Moreover, the bottom float cap 480 comprises aplurality of spaced apart fins 496 downwardly projecting from thesubstantially flat bottom surface of the circular wall 482. In oneembodiment, there are four downwardly projecting fins 496 equally spacedapart at ninety degree intervals around the centrally located aperture494 of the bottom float cap 480. In use and operation, the fins 496serve to provide an initial lift off the float assembly 340 from theinterior surface 330 of the closed bottom end 314 of the tube 312 forprecluding a vacuum seal from forming therebetween while also protectingthe elastic duckbill valve 248 under initial conditions.

Bottom float cap 480 is preferably constructed of, but not limited to, amaterial which is both biocompatible and stable for gamma irradiation.In one embodiment, the bottom float cap 480 is constructed of but notlimited to, a polystyrene or polycarbonate type of material.

Bottom Cap 500

Referring to FIGS. 37 through 39, and in another embodiment, the funneland isolation float 342 is further comprised of the bottom float cap 500which comprises a circular wall 502 comprised of a substantially flattop surface 504, a substantially flat bottom surface 506, and an outerperipheral edge 508 extending therebetween. Additionally, the bottomfloat cap 500 comprises an annular lip or projection 510 which protrudesfrom the substantially flat top surface 504 at a location adjacent theouter peripheral edge 508 of the circular wall 502 and which has atriangularly shaped cross sectional area sized to be received within acomplementally shaped annular recess 538 (FIG. 25) disposed in the floatbody 344. Furthermore, the bottom float cap 500 comprises a centrallylocated annular ledge 512 recessed below the substantially flat topsurface 504 of the circular wall 502. The centrally located annularledge 512 circumscribes a centrally located aperture 514 disposedthrough the circular wall 502. Moreover, the bottom float cap 500comprises a plurality of spaced apart cylindrically shaped legs 516downwardly projecting from the substantially flat bottom surface 506 ofthe circular wall 502. In one embodiment, there are four downwardlyprojecting cylindrically shaped legs 516 equally spaced apart at ninetydegree intervals around the centrally located aperture 514 of the bottomfloat cap 500. Each cylindrically shaped leg 516 includes a blind bore518 downwardly extending from an open end disposed in the substantiallyflat top surface 504 of the circular wall 502. In use and operation, thecylindrically shaped legs 516 serve to provide an initial lift off thefloat assembly 340 from the interior surface 330 of the closed bottomend 314 of the tube 312 for precluding a vacuum seal from formingtherebetween while also protecting the elastic duckbill valve 248 underinitial conditions. Bottom float cap 500 is preferably constructed of,but not limited to, a material which is both biocompatible and stablefor gamma irradiation. In one embodiment, the bottom float cap 500 isconstructed of, but not limited to, a polystyrene or polycarbonate typeof material.

In addition, and referring to FIGS. 40 and 41, each empty blind bore 518can serve as a carrier site for a cylindrically shaped weight 530 thatis inserted therein for the purpose of controlling the overall densityof the float assembly 340. Thus, one or more the weights 530 can becoupled with the bottom float cap 500 to provide ballast for the purposeof controlling the overall density of the float assembly 340. Thepreferred materials for the ballast or weights to adjust the specificgravity of the float assembly 340 are metal rods such as stainlesssteel. For applications of the invention to blood and bone marrowprocessing to harvest buffy coat, it is desirable to have the floatassembly 340 be tuned between the density of plasma and packed red cellsand specifically a preferred float assembly density is in the range of1.02 to 1.08 grams/cubic centimeter and most preferentially in the rangeof 1.03 to 1.07 grams/cubic centimeter. The specific density used can beoptimized for the intended therapeutic or diagnostic use in regard tothe cell composition for the device to be produced. The heavier weightwill cause an increase in red cell content and increased white bloodcell (WBC) recovery.

Referring to FIGS. 42 and 43, each cylindrically shaped weight 530 canalso be carried by each cradle structure 532 formed on the flat topsurface 484 of the bottom float cap 480 and received within one of thefour spaced apart cavities 394, 396, 398, and 400 in the bottom surface386 of the float body 344 (FIG. 28) when the bottom float cap 480 ismated with the float body 344. Thus, one or more weights 530 can becoupled to the bottom float cap 480 to provide ballast for the purposeof controlling the overall density of the float assembly 340 asdelineated above.

Furthermore, and referring to FIG. 44, cylindrically shaped weights 300can be received within blind bores 302 for fine tuning or controllingthe overall density of the float assembly 130. Thus, one or more weights300 can be coupled to the float body 134 to provide ballast for thepurpose of controlling the overall density of the float assembly 130. Inthis embodiment, the preferred materials for the weights 300 to adjustthe specific gravity of the float assembly 130 are also metal rods suchas stainless steel. Again, for applications of the invention to bloodand bone marrow processing to harvest huffy coat, it is desirable tohave the float assembly 130 be tuned between the density of plasma andpacked red cells and specifically a preferred float assembly density isin the range of 1.02 to 1.08 grams/cubic centimeter and mostpreferentially in the range of 1.03 to 1.07 grams/cubic centimeter. Thespecific density used can be optimized for the intended therapeutic ordiagnostic use in regard to the cell composition for the device to beproduced. The heavier weight will cause an increase in red cell contentand increased white blood cell (WBC) recovery.

Umbrella Valve 234

Referring to FIGS. 25, 30 through 33, and back to FIG. 14, oneembodiment of the float assembly 340 further comprises the first one wayvalve or check valve in the form of the elastic umbrella valve 234 whichis used to selectively open and close the funnel fluid passageways orapertures 450 based on a pressure differential of biological fluid onthe umbrella valve 234 thereby controlling fluid flow from the funnelshaped cavity 426 to the inverted dome shaped isolation chamber 378while precluding fluid from flowing back out of the inverted dome shapedisolation chamber 378 to the funnel shaped cavity 426 via the umbrellavalve 234. Thus, the umbrella valve 234 provides a unidirectional flowof biological fluid from the second or upper volume zone 338 above thefloat to the third volume zone or isolation zone defined by inverteddome shaped isolation chamber 378 as a function of the pressuredifferential of biological fluid on the umbrella valve 234.

More specifically, and as described above and as illustrated in FIG. 14,one embodiment of the umbrella valve 234 is comprised of the generallycircular canopy or dome 236 which, in an unstressed position, extendsgenerally perpendicularly to the centrally located stem 238. The stern238 includes the bulbous portion 240 beyond which is the tapered shaftportion 242. In one embodiment, the entire umbrella valve 234 is a onepiece, integral construction.

Referring to FIGS. 25, 30, 33, and back to FIG. 14, the umbrella valve234 is mounted to the funnel float cap 420 by securing the stem 238through the central open ended cylindrical opening 448 disposed throughthe funnel float cap 420. This is accomplished by sizing the length ofstem 238 between the canopy 236 and the bulbous portion 240 greater thanthe length of the cylindrical opening 448 and sizing the diameter of thebulbous portion greater than the diameter of the cylindrical opening 448such that when the tapered shaft portion 242 of the stem 238 is insertedinto the cylindrical opening 448 from the substantially flat bottomsurface 478 of the funnel float cap 420 and pulled or stretched awayfrom the funnel shaped upper surface 424 of the funnel float cap 420 theelastic bulbous portion 240 passes through the central open endedcylindrical opening 448 and resumes its normal shape adjacent the funnelshaped upper surface 424 of the funnel float cap 420 thereby acting asan anchor for holding the umbrella valve 234 in place. After anchoringthe umbrella valve 234 in place, the tapered shaft portion 242 may betrimmed while retaining the bulbous portion 240.

Additionally, the generally circular canopy or dome 236 includes a flatunderside contact surface 244 when in the unstressed position. Thus,when the centrally located stem 238 is stretched an axial force isexerted on the canopy or dome 236 such that the underside 244 is drawninto a normal tight, sealing contact against the substantially flatbottom surface 478 of the funnel float cap 420 for sealing the funnelfluid passageways or apertures 450.

With this construction, the elastic umbrella valve 234 provides a oneway valve which controls fluid flow into the inverted dome shapedisolation chamber 378 and precludes fluid flow out of the inverted domeshaped isolation chamber 378 via the elastic umbrella valve 234. Inparticular, the elastic umbrella valve 234 opens under a predeterminedpressure differential or cracking pressure or, in other words, when thepressure in the funnel fluid passageways or apertures 450 is greaterthan below or on an outer surface 246 of the canopy or dome 236 by apredetermined or known cracking pressure of the umbrella valve 234.Hence, when there is a positive pressure differential defined by apressure at the funnel fluid passageways or apertures 450 being greaterthan the pressure on the outer surface 246 of the canopy or dome 236 byan amount which is greater than the cracking pressure then the pressuredifferential causes the flexible canopy or dome 236 to invert or flexdownwardly, away from the substantially flat bottom surface 478 of thefunnel float cap 420 thereby permitting the biological fluid to passinto the inverted dome shaped isolation chamber 378. After the pressuredifferential resides to a point less than the cracking or openingpressure, the resilient canopy or dome 236 resumes its sealing positionunder the funnel fluid passageways or apertures 450 thereby precludingany further biological fluid from entering the inverted dome shapedisolation chamber 378.

Duckbill Valve 248

Referring to FIGS. 25, 27, 34 through 39, and back to FIG. 15, oneembodiment of the float assembly 340 further comprises the second oneway valve or check valve in the form of an elastic duckbill valve 248which is used to selectively open and close the exit passageway oraperture 380 extending between the first circular exit port 382 and thesecond circular exit port 384 based on a pressure differential ofbiological fluid on the duckbill valve 248 thereby controlling fluidflow from the inverted dome shaped isolation chamber 378 to the first orlower volume zone 336 while precluding fluid from flowing back into theinverted dome shaped isolation chamber 378 from the first or lowervolume zone 336 via the duckbill valve 248. Thus, the duckbill valve 248provides a unidirectional flow of biological fluid from the isolationchamber 378 within the float assembly 340 to the first or lower volumezone 336 as a function of the pressure differential of biological fluidon the duckbill valve 248.

In one embodiment, and as described above and as illustrated in FIG. 15,the duckbill valve 248 is comprised of the open ended hollow cylindricalportion 250 coupled to the exit passageway 170 and transitioning fromthe radially outwardly projecting annular flange portion 252 to thehollow V-shaped or converging portion 254 which terminates to theelongated outlet slit 256 defined by the pair of resilient sealing lips258. The resilient sealing lips 258 are formed to move apart to open theslit 256 for fluid to flow in one direction through the fluid passagewayaxially extending through the duckbill valve 248.

The resilient sealing lips 258 normally maintain the slit 256 in theclosed position. When fluid pressure within the hollow V-shaped orconverging portion at a location above the sealing lips 258 is greaterthan a fluid pressure below the resilient sealing lips 258 by apredefined cracking pressure of the duckbill valve 248 then theresilient sealing lips 258 spreading and the slit 256 open therebypermitting fluid to flow downwardly through the exit passageway oraperture 380 and through the axially extending fluid passageway of theduckbill valve 248. The outlet slit 256 closes when the fluid pressurebelow or on the outer surfaces of the resilient sealing lips 258 ishigher than the fluid pressure above the resilient sealing lips 258 bymore than the cracking pressure of the duckbill valve 248 therebypreventing the backflow of fluid through the duckbill valve 248.Additionally, if fluid flow stops or reverses direction, theback-pressure exerted by the fluid upon the outer surfaces of theresilient sealing lips 258 forces the lips into sealing engagementagainst one another, closing outlet slit 256 and preventing fluidbackflow.

As assembled, the annular flange portion 252 of the duckbill valve isseated between the bottom surface 386 of the float body 344 and theannular ledge 492 of the bottom float cap 480 or the annular ledge 512of the bottom float cap 500 such that the cylindrical portion 250 ofduckbill valve 248 respectively passes through the centrally locatedaperture 494 or 514 and such that the hollow V-shaped or convergingportion 254 extends respectively below the bottom float cap 480 or 500.In this arrangement, the exit passageway or aperture 380 is in opencommunication with the axially extending fluid passageway of theduckbill valve 248 and a press type fit or coupling is provide betweenthe bottom surface 386 of the float body 344, the duckbill valve 248,and either the annular ledge 492 of bottom float cap 480 or the annularledge 512 of bottom float cap 500 for maintaining a seal between theduckbill valve 248 and the float body 344.

Use and Operation

In use and operation, and referring to the drawings, the device 310follows the use and operation delineated hereinabove for device 10.Initially device 310 receives a multiple component biological fluidsample (e.g., peripheral blood, bone marrow aspirate, blood components,cord blood, apheresis blood products, lipo-aspirate, semen, urine, milk,ascites fluid, exudate or cerebrospinal fluids) by performing the stepsof removing the cap 85 from the male luer lock head 88 of the inletvalve 86, coupling a conventional needleless syringe 270 or otherdispensing device containing the biological fluid sample to the maleluer lock head 88 of the inlet valve 86, injecting or dispensing thebiological fluid sample into the device 310 from the conventionalsyringe 270 or other dispensing device, decoupling he conventionalsyringe 270 or other dispensing device from the male luer lock head 88of the inlet valve 86, and reattaching cap 85. When supplying the device310 with biological fluid, the air filter allows air to escape from theenclosure 334.

The received biological fluid sample fills the second or upper volumezone 338 above the inverted dome shaped isolation chamber 378 includingthe funnel shaped cavity 426 and flows to the first or lower volume zone336 below the float assembly 340 via the circumferential gap 356disposed between the outer circumferential surface 352 of the floatsidewall 350 and the inner circumferential surface 324 of the tube 312.The biological fluid sample is initially precluded from entering orpassing through the inverted dome shaped isolation chamber 378 by thenormally closed umbrella valve 234 and the normally closed duckbillvalve 248. Additionally, the spaced apart fins 496 of bottom float cap480 or the spaced apart cylindrical legs 516 of the bottom float cap 500provide an initial lift off the float assembly 340 from the interiorsurface 330 of the closed bottom end 314 of the tube 312 for precludinga vacuum seal from forming therebetween while also protecting theelastic duckbill valve 248 under initial conditions.

Once the biological fluid sample has been received within the enclosure334 defined by the tube 312 and tube cap 50, the device 310 is placed ina conventional centrifugation device 280 which is operated for one ormore predetermined durations at one or more predetermined speeds. In oneembodiment, and for a biological fluid sample exemplified by blood, theconventional centrifugation device 280 is operated for about 12 to about15 minutes at about 3,200 RPM. Of course, one or more time durations andone or more speeds can be empirically determined for a specificbiological fluid to be processed by the device 310 and may vary from onebiological fluid to another.

Upon initial centrifugation of the device 310, the biological fluid inthe funnel shaped cavity 426 increasingly applies a pressure on theumbrella valve 234 which results in a pressure differential on theumbrella valve 234 becoming greater than the cracking pressure of theumbrella valve 234 thereby allowing fluid to flow through one or more ofthe funnel fluid passageways or apertures 450 and into the inverted domeshaped isolation chamber 378 which begins to fill and apply a secondpressure on the duckbill valve 248 which results in a pressuredifferential on the duckbill valve 248 being greater than the crackingpressure of the duckbill valve 248 thereby allowing biological fluid toinitially flow out of the inverted dome shaped isolation chamber 378 viaexit passageway or aperture 380. At first, the biological fluid quicklyflows through the inverted dome shaped isolation chamber 378 and whenthe sample is blood the hematocrit is initially not concentrated. Withboth the umbrella valve 234 and duckbill valve 248 in an open position,the building of the column of biological fluid in the first or lowervolume zone 336 or below the float assembly 340 continues. As thiscolumn of the biological fluid builds, the device 310 provides a uniquecirculation process or density feedback process which will be clearlydelineated by using peripheral blood as an example of the biologicalfluid being processed.

Accordingly, and as the biological fluid such as peripheral bloodstratify based on density and the components build the column, the cellsthat are just below the bottom surface 484 of the bottom float cap 480or the bottom surface 506 of the bottom float cap 500 continue tostratify such that the cell density above the level of the duckbillvalve 248 is less than the cell density below the level of the duckbillvalve 248 by an amount which provides a pressure differential which isless than the cracking pressure of the duckbill valve 248 therebyresulting in the closure of the duckbill valve 248 such that over timethe reds cells accumulate at the bottom section 362 of the inverted domeshaped isolation chamber 378 and become packed and thus, there arepacked cells at the bottom section 362 of the chamber which have agreater density than the cells right below the level of the duckbillvalve 248 because at the same time that the density of red cells isincreasing at the bottom section 362 the density right below theduckbill valve 248 is decreasing because greater density cells are alsomigrating toward the bottom end 314 of the tube 312. Thus, as thisdensity increases a pressure differential builds to surpass the crackingpressure of the duckbill valve 248 which then opens thereby allowing thered cells to flow through the exit passageway or aperture 380 anddisplace the biological fluid in the first or lower zone 336 which, inturn, pushes fluid up or causes a surge of fluid up through thecircumferential gap 356 between the outer circumferential surface 352 ofthe float body sidewall 350 and the inner circumferential surface 324 ofthe tube 312. This upward fluid flow has the effect of carrying thelighter components of the biological fluid including the white bloodcells and platelets from the first or lower volume zone 336 of the tube312 back up to the second or upper volume zone 338 of the tube 312wherein these lighter components are heavier than the plasma in thesecond or upper volume zone 338 that they got circulated into so thewhite blood cells and platelets or buffy coat included therein fall backdown through the funnel shaped cavity 426, past the umbrella valve 234,and collect in the inverted dome shaped isolation chamber 378. This cellcirculation process or density feedback process continues on towards anequilibrium where the pressure differential or the density differentialof the cells above and below duckbill valve 248 is less than thecracking pressure of the duckbill valve 248 resulting in the duckbillvalve 248 having a final closure and similarly the density differentialof the cells above and below umbrella valve 234 is less than thecracking pressure of the umbrella valve 234 resulting in the umbrellavalve 234 having a final closure thereby concluding the unique cellcirculation process or density feedback process of the device 310.

Hence, this cell circulation process or density feedback process allowsmultiple chances at capturing the white cells or other target componentsof interest in the inverted dome shaped isolation chamber 378 therebyproviding higher recovery rates as compared to know prior art devices.

Following the centrifugation step, the device 310 containing theseparated and isolated biological fluid components is removed from thecentrifugation device 280 and placed on a conventional magnetic stirrer290 as diagrammatically shown in FIG. 20. The stirrer is then energizedfor activating or coacting with a magnetic stirring bar 292 locatedinside the inverted dome shaped isolation chamber 378 for stirring atleast one isolated target component within the isolation chamber 378 forincreasing a recovery rate of a subsequent harvesting step. In oneembodiment, at least the one isolated target component or fraction isstirred for about twenty seconds. Of course, stirring times may varyfrom one biological fluid to another and a specific stirring time for aspecific biological fluid can be empirically determined.

Following the stirring step, the harvesting step of at least the oneisolated target component or fraction is performed and is comprised ofthe steps of removing the cap 99 from the male luer lock head 102 of theextraction valve 100, coupling a conventional needleless syringe 272 orother extraction device to the male luer lock head 102 of the extractionvalve 100, aspirating at least the one isolated target component orfraction from the inverted dome shaped isolation chamber 378 byproviding a vacuum from the syringe 103 to the extraction passageway oraperture 404 and onto port 414 disposed in the radiused bottom section362 of the inner hemispherical surface 358 and aspirating at least theone isolated target component or fraction from the inverted dome shapedisolation chamber 378 and through the port 414 and into and through theextraction passageway or aperture 404 disposed in the float body 344,the coiled tube 124, the barb fitting 114, the L-shaped housingpassageway 62, the extraction valve 100, and finally into theconventional needleless syringe 272.

After the harvesting step is completed, the conventional needlelesssyringe 103 is decoupled from the male luer lock head 102 of theextraction valve 100 in preparation for at least one diagnostic ortherapeutic application of at least the one target component orfraction. When harvesting at least the one target component or fraction,the air filter allows air into the enclosure 48. Additionally, when thecracking pressure of the umbrella valve is reached during a harvestingstep, plasma flows into the inverted dome shaped isolation chamber 378to displace the removed volume of at least the one target component orfraction.

It should be noted that a counter weight device 282 may be used in theconventional centrifugation device 280 to offset the weight of thedevice 310 containing biological fluid as required.

Furthermore, 310 should be assembled in an environment to minimize therisk of particulate matter in the fluid path and when used for clinicalapplications, the device 310 must be sterilized and the fluid pathshould be non-pyrogenic.

Moreover, the materials used for the construction of the variousembodiments of the device for separating and isolating components of abiological fluid include plastics, rubber, metal and magnet that arebiocompatible and substantially free of any cytotoxic leachables.Preferred plastics for the construction of the tube are polystyrene orpolycarbonate. Plastics for the construction of the body of the funneland isolation float include polystyrene or polycarbonate. Preferredmaterials for the one way valves in the float are silicone rubbersstable to gamma irradiation. The preferred materials for the ballast orweights to adjust the specific gravity of the funnel assembly are metalrods such as stainless steel that are disposed in a location within thefunnel and isolation float that does not permit contact with thebiological components. Preferred magnetic stirring bars placed into theisolation chamber of the funnel and isolation float are made from amagnetic material such as neodymium magnet. The magnetic stirring barcan be a variety of shapes and sizes but should be selected so as to becompatible with the intended specific gravity of the float and to avoidthe possibility of the magnetic stirrer obstructing the flow of liquidsin the outlet apertures of the interior chamber of the float. A simplerod magnet having a size that is approximately half of the interiordiameter of the interior of the isolation chamber of the float isappropriate.

A preferred method of preparing the sample tube, sample tube cap andfunnel and isolation float is by injection molding. Alternatively, thefunnel and isolation float can be manufactured by means of tools presentin machine shop such as lathes and drills. The coiled tube used toconnect the lid to the float is preferably medical gradepolyvinylchloride. Because the float must be made with precision, it ispreferable to make the device in subcomponents to facilitate precisioninjection molding and it is preferable the subcomponents be assembledusing sonic welding devices or other reliable means.

The above delineation of the embodiments 10 and 310 of the device,including their use and operation, demonstrates the industrialapplicability of this invention,

Accordingly, it should be apparent that further numerous structuralmodifications and adaptations may be resorted to without departing fromthe scope and fair meaning of the present invention as set forthhereinabove and as described herein below by the claims.

We claim:
 1. A device for separating and isolating components of abiological fluid with centrifugation, said device comprising: anenclosure for containing a biological fluid having multiple components;a float slideably disposed within said enclosure for partitioning saidenclosure into a lower volume zone and an upper volume zone, said floathaving a central longitudinal axis; said float having an interiorisolation chamber and at least one fluid passageway providing open fluidcommunication between said upper volume zone and said interior isolationchamber; said interior isolation chamber comprising a sealing surfaceunder said at least one fluid passageway; a valve comprising a flexiblemember disposed within said interior isolation chamber, said flexiblemember comprising an upper surface facing said at least one fluidpassageway and a lower surface facing said interior isolation chamber;said flexible member configured in a unstressed state to extendgenerally perpendicularly to said central longitudinal axis of saidfloat and to close said at least one fluid passageway by providingsealing contact of said upper surface of said flexible member againstsaid sealing surface under said at least one fluid passageway; and saidflexible member configured in a stressed state to flex downwardly awayfrom said sealing surface under said at least one fluid passagewaytowards said central longitudinal axis of said float to open said atleast one fluid passageway.
 2. The device of claim 1 wherein saidunstressed state is defined as a state when fluid pressure on said uppersurface of said flexible member is less than said fluid pressure on saidlower surface of said flexible member by a predefined amount.
 3. Thedevice of claim 2 wherein said stressed state is defined as a state whenfluid pressure on said upper surface of said flexible member is greaterthan fluid pressure on said lower surface of said flexible member by apredefined amount.
 4. The device of claim 3 wherein said float furthercomprises an upper portion having a funnel shaped surface defining afunnel shaped cavity converging toward said at least one fluidpassageway and said valve wherein said valve is configured to have saidstressed and unstressed states for respectively allowing and precludingflow of biological fluid from said upper volume zone into said interiorisolation chamber of said float by way of said at least one fluidpassageway.
 5. The device of claim 4 wherein said float furthercomprises a lower portion comprising an inverted hemispherically shapedinterior surface having an upper annular edge transitioning into anupper ceiling interior surface for defining said interior isolationchamber as an inverted dome shaped isolation chamber.
 6. The device ofclaim 5 wherein said upper ceiling interior surface of said interiorisolation chamber comprises said sealing surface under said at least onefluid passageway.
 7. The device of claim 6 wherein said sealing surfaceunder said at least one fluid passageway is a substantially flatsurface.
 8. The device of claim 7 wherein said upper surface of saidflexible member of said valve is a substantially flat surface configuredin said unstressed state to seal said at least one fluid passageway byproviding sealing contact against said substantially flat surface ofsaid sealing surface under said at least one fluid passageway.
 9. Thedevice of claim 8 wherein said float includes an aperture means in opencommunication with said interior isolation chamber of said float forharvesting at least one isolated component from said interior isolationchamber of said float.
 10. A device for separating and isolatingcomponents of a biological fluid with centrifugation, said devicecomprising: an enclosure for containing a biological fluid havingmultiple components; a float slideably disposed within said enclosureand comprising an interior isolation chamber; said float partitioningsaid enclosure into a lower volume zone and an upper volume zone; saidfloat comprising a first valve through which the biological fluid flowsfrom said upper volume zone and into said interior isolation chamber ofsaid float as a function of a first pressure differential of biologicalfluid on said first valve; and said float having a second valve throughwhich the biological fluid flows out of said interior isolation chamberof said float and into said lower volume zone as a function of a secondpressure differential on said second valve.
 11. The device of claim 10wherein said float further comprises an upper portion having a funnelshaped surface defining a funnel shaped cavity converging toward saidfirst valve for promoting said flow of biological fluid into saidinterior isolation chamber as said function of said first pressuredifferential of biological fluid on said first valve.
 12. The device ofclaim 11 wherein said float further comprises a lower portion comprisingan inverted hemispherically shaped interior surface having an upperannular edge transitioning into an upper ceiling interior surface fordefining said interior isolation chamber as an inverted dome shapedisolation chamber.
 13. A device for separating and isolating componentsof a biological fluid with centrifugation, said device comprising: anenclosure for containing a biological fluid having multiple components;a float slideably disposed within said enclosure and having an interiorisolation chamber; said float partitioning said enclosure into a lowervolume zone and an upper volume zone; a first valve for allowing a flowof biological fluid from said upper volume zone and into said interiorisolation chamber of said float as a function of a first pressuredifferential of biological fluid on said first valve; and a second valvefor providing a flow of biological fluid out of said interior isolationchamber of said float and into said lower volume zone as a function of asecond pressure differential of biological fluid on said second valveWherein under device centrifugation said first valve and said secondvalve initially allow components of the biological fluid to flow throughsaid interior isolation chamber and subsequently close to seal saidinterior isolation chamber as said function of said first pressuredifferential of biological fluid on said first valve and as saidfunction of said second pressure differential of biological fluid onsaid second valve for isolating at least one target component of thebiological fluid within said interior isolation chamber of said float.14. The device of claim 13 wherein said float further comprises an upperportion having a funnel shaped surface defining a funnel shaped cavityconverging toward said first valve for promoting said flow of biologicalfluid into said interior isolation chamber as said function of saidfirst pressure differential of biological fluid on said first valve. 15.The device of claim 14 wherein said float further comprises a lowerportion comprising an inverted hemispherically shaped interior surfacehaving an upper annular edge transitioning into an upper ceilinginterior surface for defining said interior isolation chamber as aninverted dome shaped isolation chamber.